1. Field of the Invention
This invention relates to the control of viral infections of fish and more specifically to vaccines for controlling outbreaks of infectious pancreatic necrosis virus infections in fish populations. 2. General Discussion of the Background
It is estimated that fish diseases cost twenty to thirty cents for each dollar spent rearing fish in the United States. Although fish pathogens include fungal, protozoan and bacterial agents, it is the viral diseases that most concern hatchery managers and fisheries scientists because they are largely uncontrollable. There are no antibiotic agents that work effectively against viruses in fish.
Infectious pancreatic necrosis (IPN), a contagious viral disease in fish, causes morbidity and mortality in juvenile rainbow trout, Atlantic salmon and brook trout. The virus, infectious pancreatic necrosis virus (IPNV), has been isolated from both salmonid and non-salmonid species of fish lacking pathogenic symptoms. In survivors of an IPNV epizootic, the virus persists and can cause severe growth retardation in individual fish exhibiting virus persistence. McKnight and Roberts, Br. Vet. J. 132: 76-86, 1976. In smolts, the virus produces considerable necrosis or inflammation of the pancreas. The virus is capable of infecting a number of different hosts and has a worldwide presence. Pilcher and Fryer, Crit. Rev. Microbiol. 7:287-364, 1980.
IPN disease in a brook trout hatchery was first reported in 1941. McGonigle, Trans. Am. Fish. Soc. 70:297, 1941. In 1960, the viral nature of the disease was confirmed. Wolf et al., Proc. Soc. Exp. Biol. Med. 104:105-110, 1960. Since that time there have been isolations of the virus in a variety of fish species throughout the world, including various trout and salmon species, carp, perch, pike, eels and char, as well as mollusks and crustaceans. Acute disease has been reported primarily in a limited number of salmonid species, such as trout and salmon. Many of these species are of growing economic importance and since mortalities range as high as 90 percent, Pilcher and Fryer, Crit. Rev. Microbiol. 7:287-364, 1980, an IPN outbreak in a hatchery can be an economic disaster for the aquaculturist.
Young fish (two- to four-month old) appear to be the most susceptible to IPNV infection, resulting in high mortality. Wolf et al., U.S. Dept. Int. Bur. Sport Fish. and Wildlife, Fish Disease Leaflet 1:14, 1966; Frantsi and Savan, J. Wildlife Dis. 7:249-255, 1971. In trout, IPN usually attacks young fry about five to six weeks after their first feeding. The affected fish are darker than usual, have slightly bulging eyes and often have swollen bellies. At the beginning of an outbreak, large numbers of slow, dark fry are seen up against water outflows, and fish are seen "shivering" near the surface. The shivering results from a characteristic symptom of the disease, a violent whirling form of swimming in which the fish rotate about their long axis. If the affected fish are opened up, a characteristic white mucus is seen in the stomach. The pancreas appears to be the primary target organ for the virus, with the pancreatic fat cells or Islets of Langerhans being unaffected. McKnight and Roberts, Br. Vet. J. 132:76-86, 1976. The intestine is the only organ besides the pancreas where viral lesions are consistently found.
After an IPN outbreak, the surviving fish generally become carriers of the virus. For example, trout that are carriers of the virus are a serious problem for the aquaculture industry because the only control method currently available for eliminating the virus in carrier fish is complete destruction of these fish. Several factors, including age, species and water temperature, appear to influence the severity of infection and the subsequent establishment of the carrier state. Surviving carriers shed infectious IPNV which is detectable in fecal and sex products of the fish for the remainder of their lifetime. Billi and Wolf, J. Fish. Res. Bd. Can. 26:1459-1465, 1969; Yamamoto, Can. J. Micro. 21:1343-1347, 1975; Reno et al., J. Fish. Res. Bd. Can. 33:1451-1456, 1978.
The persistence of the virus in carrier fish appears to be the result of continued virus production by a small number of infected cells in certain organs. Only a few cells in the infected organs are capable of registering as infectious centers that actually produce virus. Hedrick, Ph.D. Thesis, "Persistent Infections of Salmonid Cell Lines With Infectious Pancreatic Necrosis Virus: A Model for the Carrier State in Trout," Oregon State University, 1980.
IPNV is a birnavirus whose genome consists of two segments of double-stranded RNA. Dobos, Nucl. Acids Res. 3:1903-1919, 1976; Dobos, J. Virol. 21:242-258, 1977. The viral genome is contained within a non-enveloped icosahedral capsid approximately 60 nm in diameter. The larger "A" segment has a molecular weight of 2.5.times.10.sup.6 Daltons (Da) and encodes at least three proteins whose order in the genome from the N (5') terminus is .beta.(VP2) (approximately 54 kDa, major capsid protein) - .gamma..sub.2 (NS) (approximately 27.5 kDa, nonstructural protein having proteolytic activity) - .gamma..sub.1 (VP3) (approximately 31 kDa, minor capsid protein), as shown in FIG. 1. Chang et al., Can. J. Microbiol. 24:19-27, 1978; Huang et al., J. Virol. 60:1002-1011, 1986. These proteins are encoded on a single mRNA within the infected cell. Mertens and Dobos, Nature 297:243-246, 1982. Sequence analysis has indicated that there is one continuous open reading frame for this viral RNA segment. Duncan and Dobos, Nucleic Acids Res. 14:5934-5935, 1986.
Currently, there are no methods for controlling IPN except the destruction of infected stocks and the decontamination of hatchery facilities. Despite a great deal of interest in developing a vaccine for IPNV and investigation of a variety of approaches, little progress has been made toward a truly practical vaccine. One approach has been the use of attenuated viral strains. See, Dorson, Abstract, International Conference on IPNV, Taloires, France, 1982. Unfortunately, the attenuated strains either fail to infect the fish or fail to induce protection. Strains with low virulence have been tested as vaccines for more virulent strains, but mortality from the vaccinating strain was either too high or protection was only moderate. Hill et al., "Studies of the Immunization of Trout Against IPN," in Fish Diseases, Third COPRAQ Session (W. Ahne, ed.), NY, pp. 29-36, 1980. Major unsolved requirements associated with the use of live, attenuated virus vaccines include: the requirement for stability of attenuation and the development of appropriate tests to assess this stability; the requirement that the virus should not establish persistent infection in the host; and the requirement that the virus should exhibit low communicability in field trials.
Use of killed virus as vaccines has also been tried. For example, if formalin-inactivated virus is injected intraperitoneally into four week post-hatch fry, the fish become immunized. Dorson, J. Virol. 21:242-258, 1977. However, neither immersion of the fish into a liquid suspension of killed virus nor oral administration thereof was effective. The main problem with using killed virus is the lack of a practical method for administration of the vaccine. Injection is impractical for large numbers of immature fish. Some investigators have suggested that the uptake of viral antigen by immersion might be improved if the virus was disrupted into smaller, sub-viral components, but viral disruption methods have resulted in loss of antigenicity. Hill et al., 1980 (supra); Hill and Way, "Serological Classification of Fish and Shellfish Birnaviruses," Abstract, First International Conference of the European Association of Pathology, Plymouth, England, 1983.
The preparation of a killed virus vaccine is very expensive. In addition, both attenuated and killed virus vaccines must be extensively purified in order to prevent cellular contamination. All of these requirements add to the cost of developing and preparing a viral vaccine by conventional methods. For fish hatcheries that operate o relatively thin profit margins, the additional expense of such vaccines is prohibitive.
Accordingly, there remains no known practical method of immunizing fish against IPNV infection, despite the need for such a method. Hence, there is a need for a method for: (1) producing sufficient quantities of a non-infectious IPNV immunogen that can be simultaneously administered to large numbers of fish; (2) a method for producing such viral immunogen in large quantities at a reasonable cost; (3) producing such viral antigen that is completely free of viable virus; and (4) administering such viral antigen to fish as early in their development cycle as possible.